This invention pertains to recombinant genes coding for oleosin proteins in cacao and to the polypeptides encoded by said genes. In particular, the present invention relates to the use of such genes and gene products for the manufacture of emulsions and flavor.
In a variety of different plants, such as e.g. in soybean, rapeseed or sunflower oily components that are insoluble in water, are stored in subcellular structures termed “oil bodies”. The oil stored in these particles form cellular food reserves that may be mobilized quickly when large increases in cellular metabolism are required, such as during seed germination or pollen tube growth. Most plant seeds contain stored TAG's (triacylglycerols) as food reserves for germination and post germination growth, although the level of TAG's stored in seeds varies between different plants.
Intracellular oil bodies of seeds are generally between 0.5 and 2 μM in diameter (Tzen et al., Plant Physiol. 101 (1993), –276) and are considered to be composed of a matrix of TAG's surrounded by a phospholipid layer and associated with a set of different proteins, that are called oil body proteins or oleosins. The function of said oleosins is deemed to reside in the maintenance of the oil reserves of seeds and pollen in small stable droplets providing a high surface to volume ratio which facilitates the rapid conversion of the TAG's into free fatty acids via lipase mediated hydrolysis at the oil body surface.
Genomic clones encoding oleosins have been isolated for two species, namely maize (Browman et al., J. Biol. Chem. 265 (1987), 11275–11279) and carrot (Hatzopoulos et al., Plant Cell 2 (1990), 457–467). Moreover, from the cultivated oilseed Brassica napus cDNA clones could be obtained and the genomic organisation of the corresponding gene could be verified (Murphy et al., Biochem. Biophys. Acta 1088 (1991), 86–94). However, not any plant is presumed to make use of such oleosins and for cacao it was generally held that no such genes/proteins are present (Leprince et al., Planta 204 (1998), 109–119).
Most of the plant seed oil bodies and/or oleosins analyzed to date have been derived from seeds that undergo drying during maturation and can be stored safely for long periods under dry, low temperature conditions (“orthodox” seeds). To this end, genomic clones encoding oleosins have been isolated for two species, namely maize (Browman et al., J. Biol. Chem. 265 (1987), 11275–11279) and carrot (Hatzopoulos et al., Plant Cell 2 (1990), 457–467). Moreover, Murphy et al. report in Biochem. Biophys. Acta 1088 (1991), 86–94 the isolation of a cDNA clone and the genomic organisation of oleosin in the cultivated oilseed Brassica napus. 
In addition, studies have been carried out on oleosin proteins of two other groups of seeds. Seeds that do not undergo desiccation during late maturation and are usually killed at a high water content and low temperatures (recalcitrant seeds; e.g. cacao and red oak) and seeds that do undergo desiccation, but are sensitive to storage at temperatures below 0° C. (“intermediate” seeds; coffee and neem) (Leprince et al. (1998) Planta 204, 109–119.). The data presented in this report lead to the conclusion that the seeds of red oak had very low levels of oleosin proteins while cacao did not seem to have any oleosin proteins at all. In contrast thereto, “intermediate” seeds were shown to have both oil bodies by electron microscopy and levels of oleosins similar to that observed in “orthodox” seeds, such as the rape seed Brassica napa. 
The known oleosins turned out to be small alkaline proteins having an average weight of about 15 to 26 kDa and exhibiting an unusually long central hydrophobic region (about >70 amino acids). In an intact oil body within the cell this hydrophobic region is deemed to reside within the TAG matrix and anchor the oleosin in the oily central matrix. The N-terminal region of known oleosin proteins have been found to be rather diverse both in sequence and length.
Cacao is an important raw material for manufacturers of confectionery and other products, for e.g. chocolate. It is known that during fermentation of cacao the existing protease activity in the cacao seed results in the formation of an increased level of cacao flavor precursors, such as hydrophilic peptides and hydrophobic amino acids, which contribute significantly to the typical flavor the consumer knows as the cacao flavor (Mohr, W. W., Landschreiber, E., and Severin, T., (1976) Fett. Wissenschaft. Technologie Vol 78 88–95; Voigt, J., Biehl, B., Heinrichs, H., Kamaruddin, S., Gaim Marsoner, G., and Hugi, A. 1994 Food Chemistry 49, 173–180). This increase of flavor precursor peptides and hydrophobic amino acids is dependent on the proteolytic activity within the seed during the fermentation process and on the amount of proteins containing these precursor peptides and hydrophobic amino acids. The progress of the fermentation reaction has to be carefully monitored so that the desired cacao flavor precursors will be eventually obtained. Also, the raw materials have to be evaluated for flavor potential, since cacao seeds deficient in an appropriate amount of proteins containing flavor precursor peptides and hydrophobic amino acids will result in a fermented material deficient in cacao flavor precursors. Consequently, there is a need in the art to provide cacao raw material that constantly has a sufficient amount of cacao flavor precursor peptides and hydrophobic amino acids.